Diabetes mellitus is a complex disease which affects up to 5% of the human population. Its primary clinical symptom is abnormally high levels of plasma glucose. The regulatory hormones that control glucose homeostasis are insulin and glucagon, both hormones only expressed in islet cells of the pancreas. In order to understand why the insulin gene is only expressed in islet cells, it is necessary to develop a cell-free system to identify all the necessary components required for its expression. Once these components are identified, one can then study their mechanism of action. In the last few years, we have identified the enhancer element necessary for the expression of the rat insulin II gene and have characterized four transcription factors essential for the function of this enhancer. We have recently isolated three genes (BETA1, BETA2 and Rip-1) encoding some of these proteins. One of these genes, BETA2, is a tissue-specific basic- helix-loop-helix protein which binds to the important regulatory element E box (also called RIPE3a, ICE, Nir or Far box). In this proposal, we propose to characterize these transcription factors, studying their role in insulin gene expression as well as their role in the islet cell development. We will address this question using biochemical, molecular, biological, developmental and genetic approaches. We will isolate and characterize genes encoding all players involved in the synergistic activation of the rat insulin II gene. We will also study the developmental regulation of these important factors by in situ hybridization, by immunocytochemistry, and by transgenic animals expressing the reporter, beta-gal, under the control of transacting factor promoters. Finally, using gene knockout and transgenic technologies, we will study their role in islet cell development. It is expected that the results derived from this project will be relevant to the better understanding of insulin gene regulation and islet cell formation. In addition, the following proposal should also be pertinent in elucidating a more precise hypothesis for the regulation of gene expression during differentiation, development, and cancer metastasis.